臺灣博碩士論文加值系統

CD151是一種膜蛋白，屬於tetraspanin 成員中的一員。目前CD151被發現可以調控細胞的附著能力與運動能力，並且與腫瘤的惡性化有高度的相關性。然而，卻沒有任何研究針對腫瘤細胞與正常細胞的CD151表現量作探討。在腫瘤組織中，CD151被認為可能扮演著腫瘤轉移抑制者的角色，而缺氧則是可以促進腫瘤的轉移。因此我們假設在缺氧的情況下所引發的腫瘤轉移，可能是缺氧壓力藉由負調控CD151的表現量而抑制了CD151的功能。在我們的研究中使用配對的大腸直腸檢體，也發現了在腫瘤部位的CD151表現量低於配對的正常組織，同時也觀察到缺氧誘導因子-1�悁b腫瘤部位的高度表現。利用了生物資訊方法分析之後，發現在CD151基因的驅動子區域有缺氧反應序列的存在。而利用大腸直腸癌細胞株處以缺氧或是化學性模擬缺氧處理之後，可以觀察到隨著缺氧誘導因子-1�悛穛{上升，CD151表現量明顯下降。而在驅動子活性分析的實驗中，也發現CD151基因的缺氧反應序列在缺氧環境下其轉錄活性會下降，確認了在缺氧環境中CD151基因會受到缺氧誘導因子-1�悛滬t調控。更進一步的實驗證實，缺氧處理之後CD151表現量下降同時也引起細胞的附著能力的降低，使得細胞對於胞外基質貼附變差。這樣的過程可能是促使腫瘤細胞在缺氧的壓力之下，易於離開胞外基質並且更容易轉移的準備步驟。總結以上的發現，我們證明了缺氧會透過缺氧誘導因子-1�悝磻薡D151的表現，並且使得腫瘤細胞附著能力變差，進而可能增加了細胞從基底膜的分離而使腫瘤轉移更容易發生。

CD151 is a member of tetraspanin family that plays important roles in cell adhesion and motility. However, the regulation of CD151 expression in normal and cancerous cells has not yet revealed. In cancer tissue, it is suggested that CD151 acts as a metastasis-suppressor while hypoxia has been shown to be a factor of causing metastasis. We hypothesize that hypoxia-induced cancer cell metastasis may be mediated via deregulation of CD151. Indeed, colon cancer cells expressed less CD151 than its normal counterpart, which is inversely correlated with the expression of hypoxia-inducible factor-1�� (HIF-1��). By using bioinformatic approach, we identified a putative hypoxia response element (HRE) in the promoter region of human CD151 gene. Treatment of colon cancer cells with hypoxia or chemicals that are known to increase accumulation of HIF-1�� resulted in decreased CD151 expression. Promoter activity assay further demonstrated that the predicted putative HRE was pivotal for HIF-1��-dependent downregulation of CD151 expression. Furthermore, inhibition of CD151 expression by hypoxia resulted in decreased cancer cell adhesion to laminin-coated matrix, a process that enables cancer cell to detach from its surrounding matrix and be ready for metastasis. In conclusion, we demonstrated that hypoxia downregulates the expression of CD151 thus enhances the detachment of cancer cells from its basement membrane protein, an important first step for metastasis.

目錄………………………………………………………………… 1
圖錄………………………………………………………………… 3
中文摘要…………………………………………………………… 4
Abstract…………………………………………………………… 5
緒論………………………………………………………………… 6
實驗材料與方法…………………………………………………… 15
材料: ……………………………………………………………… 15
大腸直腸癌病人腫瘤組織與正常組織的收集…………………… 15
實驗方法: ………………………………………………………… 15
組織蛋白質萃取…………………………………………………… 15
免疫組織染色……………………………………………………… 16
免疫組織染色定性分析…………………………………………… 17
細胞培養…………………………………………………………… 18
缺氧處理…………………………………………………………… 18
萃取細胞蛋白質…………………………………………………… 19
蛋白質濃度分析…………………………………………………… 19
西方轉漬法………………………………………………………… 20
細胞轉殖…………………………………………………………… 21
螢光酵素分析……………………………………………………… 22
缺氧對細胞附著影響試驗………………………………………… 23
細胞附著能力試驗………………………………………………… 23
細胞連結試驗……………………………………………………… 25
統計分析…………………………………………………………… 25
結果………………………………………… ………………………27
A. CD151在腫瘤組織的表現量低於正常組織 27
B. CD151的表現量與缺氧誘導因子-1�悛漯穛{量為負相關 28
C. 缺氧處理大腸直腸癌細胞株SW480促使CD151表現量下降 28
D. 缺氧處理影響在轉錄層次來降低CD151的mRNA 29
E. 缺氧可以透過缺氧誘導因子-1�挼梇婆D151的轉錄 30
F. 缺氧壓力使細胞貼附數目降低 30
G. 缺氧所導致的CD151下降減少了細胞的附著能力 31
H. 缺氧所導致的CD151下降減少了細胞的連結能力 33
討論………………………………………… 34
參考文獻………………………………………… 40
附圖………………………………………… 45
附錄: 溶液與藥品的配置………………………………………… 56

1.Brown, J.M. and A.J. Giaccia, The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res, 1998. 58(7): p. 1408-16.
2.Harris, A.L., Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer, 2002. 2(1): p. 38-47.
3.Bindra, R.S. and P.M. Glazer, Genetic instability and the tumor microenvironment: towards the concept of microenvironment-induced mutagenesis. Mutat Res, 2005. 569(1-2): p. 75-85.
4.Seimiya, H., et al., Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells. Biochem Biophys Res Commun, 1999. 260(2): p. 365-70.
5.Ke, Q. and M. Costa, Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol, 2006. 70(5): p. 1469-80.
6.Semenza, G., Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol, 2002. 64(5-6): p. 993-8.
7.Tian, H., S.L. McKnight, and D.W. Russell, Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev, 1997. 11(1): p. 72-82.
8.Ema, M., et al., A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A, 1997. 94(9): p. 4273-8.
9.Jiang, B.H., et al., Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem, 1996. 271(30): p. 17771-8.
10.Brahimi-Horn, C., N. Mazure, and J. Pouyssegur, Signalling via the hypoxia-inducible factor-1alpha requires multiple posttranslational modifications. Cell Signal, 2005. 17(1): p. 1-9.
11.Lando, D., et al., FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev, 2002. 16(12): p. 1466-71.
12.Tanimoto, K., et al., Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein. Embo J, 2000. 19(16): p. 4298-309.
13.Arendt, C.S. and M. Hochstrasser, Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly. Embo J, 1999. 18(13): p. 3575-85.
14.Zhong, H., et al., Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res, 1999. 59(22): p. 5830-5.
15.Trastour, C., et al., HIF-1alpha and CA IX staining in invasive breast carcinomas: prognosis and treatment outcome. Int J Cancer, 2007. 120(7): p. 1451-8.
16.Hutchison, G.J., et al., Hypoxia-inducible factor 1alpha expression as an intrinsic marker of hypoxia: correlation with tumor oxygen, pimonidazole measurements, and outcome in locally advanced carcinoma of the cervix. Clin Cancer Res, 2004. 10(24): p. 8405-12.
17.Sivridis, E., et al., Association of hypoxia-inducible factors 1alpha and 2alpha with activated angiogenic pathways and prognosis in patients with endometrial carcinoma. Cancer, 2002. 95(5): p. 1055-63.
18.Aebersold, D.M., et al., Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res, 2001. 61(7): p. 2911-6.
19.Mizukami, Y., et al., Hypoxia-inducible factor-1-independent regulation of vascular endothelial growth factor by hypoxia in colon cancer. Cancer Res, 2004. 64(5): p. 1765-72.
20.Semenza, G.L., Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003. 3(10): p. 721-32.
21.Zhang, X.A., et al., Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis. Mol Biol Cell, 2002. 13(1): p. 1-11.
22.Hasegawa, H., et al., Assignment of SFA-1 (PETA-3), a member of the transmembrane 4 superfamily, to human chromosome 11p15.5 by fluorescence in situ hybridization. Genomics, 1997. 40(1): p. 193-6.
23.Sincock, P.M., et al., PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci, 1999. 112 ( Pt 6): p. 833-44.
24.Yauch, R.L., et al., Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. Mol Biol Cell, 1998. 9(10): p. 2751-65.
25.Charrin, S., et al., Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation. FEBS Lett, 2002. 516(1-3): p. 139-44.
26.Dunphy, J.T. and M.E. Linder, Signalling functions of protein palmitoylation. Biochim Biophys Acta, 1998. 1436(1-2): p. 245-61.
27.Hemler, M.E., Specific tetraspanin functions. J Cell Biol, 2001. 155(7): p. 1103-7.
28.Zhang, X.A., A.L. Bontrager, and M.E. Hemler, Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins. J Biol Chem, 2001. 276(27): p. 25005-13.
29.Hasegawa, H., et al., SFA-1/PETA-3 (CD151), a member of the transmembrane 4 superfamily, associates preferentially with alpha 5 beta 1 integrin and regulates adhesion of human T cell leukemia virus type 1-infected T cells to fibronectin. J Immunol, 1998. 161(6): p. 3087-95.
30.Yanez-Mo, M., et al., Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions. J Cell Biol, 1998. 141(3): p. 791-804.
31.Testa, J.E., et al., Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis. Cancer Res, 1999. 59(15): p. 3812-20.
32.Takeda, Y., et al., Deletion of tetraspanin Cd151 results in decreased pathologic angiogenesis in vivo and in vitro. Blood, 2007. 109(4): p. 1524-32.
33.Sterk, L.M., et al., The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes. J Cell Biol, 2000. 149(4): p. 969-82.
34.Wright, M.D., et al., Characterization of mice lacking the tetraspanin superfamily member CD151. Mol Cell Biol, 2004. 24(13): p. 5978-88.
35.Sachs, N., et al., Kidney failure in mice lacking the tetraspanin CD151. J Cell Biol, 2006. 175(1): p. 33-9.
36.Karamatic Crew, V., et al., CD151, the first member of the tetraspanin (TM4) superfamily detected on erythrocytes, is essential for the correct assembly of human basement membranes in kidney and skin. Blood, 2004. 104(8): p. 2217-23.
37.Cowin, A.J., et al., Wound healing is defective in mice lacking tetraspanin CD151. J Invest Dermatol, 2006. 126(3): p. 680-9.
38.Serru, V., et al., Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions. Biochem J, 1999. 340 ( Pt 1): p. 103-11.
39.Kazarov, A.R., et al., An extracellular site on tetraspanin CD151 determines alpha 3 and alpha 6 integrin-dependent cellular morphology. J Cell Biol, 2002. 158(7): p. 1299-309.
40.Chattopadhyay, N., et al., alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion. J Cell Biol, 2003. 163(6): p. 1351-62.
41.Winterwood, N.E., et al., A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5. Mol Biol Cell, 2006. 17(6): p. 2707-21.
42.Stipp, C.S. and M.E. Hemler, Transmembrane-4-superfamily proteins CD151 and CD81 associate with alpha 3 beta 1 integrin, and selectively contribute to alpha 3 beta 1-dependent neurite outgrowth. J Cell Sci, 2000. 113 ( Pt 11): p. 1871-82.
43.Tokuhara, T., et al., Clinical significance of CD151 gene expression in non-small cell lung cancer. Clin Cancer Res, 2001. 7(12): p. 4109-14.
44.Hashida, H., et al., Clinical significance of transmembrane 4 superfamily in colon cancer. Br J Cancer, 2003. 89(1): p. 158-67.
45.Ang, J., et al., CD151 protein expression predicts the clinical outcome of low-grade primary prostate cancer better than histologic grading: a new prognostic indicator? Cancer Epidemiol Biomarkers Prev, 2004. 13(11 Pt 1): p. 1717-21.
46.Gesierich, S., et al., Colocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: impact on cell motility. Clin Cancer Res, 2005. 11(8): p. 2840-52.
47.Kohno, M., et al., CD151 enhances cell motility and metastasis of cancer cells in the presence of focal adhesion kinase. Int J Cancer, 2002. 97(3): p. 336-43.
48.Chometon, G., et al., Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cells. Exp Cell Res, 2006. 312(7): p. 983-95.
49.Lu, H., et al., Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1. J Biol Chem, 2005. 280(51): p. 41928-39.
50.Metzen, E., et al., Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases. Mol Biol Cell, 2003. 14(8): p. 3470-81.
51.Wouters, B.G. and J.M. Brown, Cells at intermediate oxygen levels can be more important than the "hypoxic fraction" in determining tumor response to fractionated radiotherapy. Radiat Res, 1997. 147(5): p. 541-50.
52.Teicher, B.A., J.S. Lazo, and A.C. Sartorelli, Classification of antineoplastic agents by their selective toxicities toward oxygenated and hypoxic tumor cells. Cancer Res, 1981. 41(1): p. 73-81.
53.Sierra, A., Metastases and their microenvironments: linking pathogenesis and therapy. Drug Resist Updat, 2005. 8(4): p. 247-57.
54.Erler, J.T., et al., Lysyl oxidase is essential for hypoxia-induced metastasis. Nature, 2006. 440(7088): p. 1222-6.
55.Kagan, H.M. and W. Li, Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem, 2003. 88(4): p. 660-72.
56.Erler, J.T. and A.J. Giaccia, Lysyl oxidase mediates hypoxic control of metastasis. Cancer Res, 2006. 66(21): p. 10238-41.
57.Sogawa, K., et al., Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation. Proc Natl Acad Sci U S A, 1995. 92(6): p. 1936-40.
58.Chapman-Smith, A., J.K. Lutwyche, and M.L. Whitelaw, Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators. J Biol Chem, 2004. 279(7): p. 5353-62.
59.Swanson, H.I., W.K. Chan, and C.A. Bradfield, DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins. J Biol Chem, 1995. 270(44): p. 26292-302.
60.Wang, G.L., et al., Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A, 1995. 92(12): p. 5510-4.
61.Wood, S.M., et al., The role of the aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells. J Biol Chem, 1996. 271(25): p. 15117-23.
62.Chen, K.F., et al., Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha. Nucleic Acids Res, 2005. 33(16): p. 5190-8.
63.Pal, S., et al., mSin3A/histone deacetylase 2- and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad. Mol Cell Biol, 2003. 23(21): p. 7475-87.